Hepatitis B virus X protein-mediated upregulation of miR-221 activates the CXCL12-CXCR4 axis to promote NKT cells in HBVrelated hepatocellular carcinoma

Both hepatitis B virus X protein(HBx)and microRNA-221(miR-221)have been implicated in the development of hepatitis B virus(HBV)-related hepatocellular carcinoma(HCC).The present study demonstrates that HBx promotes HCC cell proliferation via the C-X-C motif chemokine ligand 12-C-X-C chemokine receptor type 4(CXCL12-CXCR4)axis.We predict that HBx/miR-221-mediated CXCL12/CXCR4 signaling induces NKT cells to promote HBV-related HCC.Methods:After miR-221 mimic,miR-221 mimic negative control,miR-221 inhibitor,miR-221 inhibitor negative control were transfected into cells,the expression of CXCL12 and miR-221 was detected by qPCR and western blot.Then we constructed a stable HBV-HCC cell line.HBV-HCC cells were injected into the nude mice,thus a HBV-HCC mouse model was constructed.Q-PCR and western blot were used to detect the expression of HBx,miR-221,CXCL12 and CXCR4 in tumor tissues.The expression of CXCL12 was detected by immunohistochemistry,and the expression of CXCR4,CD3 and CD56 was detected by immunofluorescence.The levels of CXCL12,IL-2 and TNF-αin serum of mice were detected by ELISA.Sixty-one patients with HBV-related HCC,61 patients with HBV-related cirrhosis,61 patients with chronic hepatitis B(CHB)and 30 healthy people were enrolled.CXCL12,cytokine levels,and clinicopathological parameters were tested.Results:Hepatitis B virus X protein upregulates the expression of miR-221 and CXCL12 in lentivirus(LV5)-HBx-transfected HepG2 cells.HBx protein promotes HepG2 cell proliferation in vitro.HBx protein promoted tumor growth via the miR-221/CXCL12/CXCR4 pathway in a mouse tumor model.HBx protein upregulated natural killer T cell expression via the CXCR4/CXCL12 pathway to promote tumor growth.The data demonstrated a positive correlation between CXCL12 concentration with Cre levels and Child-Pugh scores.CXCL12 had an inferior diagnostic efficiency compared to IL-2 and IL-6 for HBV-related HCC.Conclusions:We present evidence that HBx/miR-221-mediated CXCL12/CXCR4 signaling induces NKT cells to promote HBV-related HCC.

Construction of an immune-related prognostic model to predict prognosis and immunotherapy in liver cancer patients with hepatitis B virus-infected

Background:Hepatocellular carcinoma(HCC)appears to be strongly associated with immune-related genes.However,immune-related genes are not well understood as a prognostic marker in HCC caused by the hepatitis B virus(HBV).The purpose of this study was to investigate the prognostic significance of immune-related genes in HBV-infected HCC.Methods:Gene expression data from 114 HBV-infected HCC and 50 normal tissues were integrated into The Cancer Genome Atlas.Differentially expressed immune-associated genes were analyzed to identify immune-associated differential genes associated with overall survival.Least Absolute Shrinkage and Selection Operator and multivariate Cox regressions were used to constructing immunoprognostic models.An independent prognostic factor analysis using multiple Cox regressions was also performed for HBV-infected HCCs.Immunocorrelation analysis markers and immune cell infiltration were also investigated.Results:We found 113 differentially expressed immune-associated genes.Immune-related differential genes were significantly correlated with the overall survival of HCC patients.We constructed an immune-based prognostic model using multivariate Cox regression analysis including seven immune-related genes.According to further analysis,immune-related prognostic factors may serve as independent prognostic indicators in the clinical setting.There is also evidence that the 7-gene prognostic model reflects the tumor immune microenvironment as a result of the risk score model and immune cell infiltration.Conclusions:As a result of our study,we screened immune-related genes for prognosis in HBV-infected HCC and developed a novel immune-based prognostic model.The research not only provides new prognostic biomarkers but also offers insight into the tumor immune microenvironment and lays the theoretical groundwork for immunotherapy.

Expression of hepatitis B virus genes in early embryonic cells originated from hamster ova and human spermatozoa transfected with the complete viral genome

Aim： To detect the expression of hepatitis B virus （HBV） genes （HB S and C genes） in early embryonic cells after introducing motile human sperm carrying HBV DNA into zona-free hamster oocytes via the in vitro fertilization （IVF） technique. Methods： Human sperm-mediated HBV genes were delivered into zona-free hamster oocytes by the IVF method. Polymerase chain reaction （PCR） was used to detect HB S and pre-Core/Core （pre-C/C） coding genes both in one- and two-cell embryos. Reverse transcription-PCR （RT-PCR） analysis was used to study the expression of the two genes. Fluorescence in situ hybridization （FISH） analysis using the full-length HBV DNA as the hybridization probe was performed to confirm the integration of viral DNA in the host embryonic genome. Results： Both HB S and pre-C/C coding genes are present and transcribed in one- and two-cell embryos originated from hamster ova IVF with human spermatozoa carrying HBV DNA sequences. Conclusion： Sperm-mediated HBV genes are able to replicate and express themselves in early embryonic cells. These results provide direct evidence that HBV DNA could transmit vertically to the next generation via the male germ line.

Preliminary study on the production of transgenic mice harboring hepatitis B virus X gene

AIM To establish transgenic mice lineage xharboring hepatitis B virus X gene and to provide an efficient animal model for studying the exact role of the HBx gene in the process of hepatocarcinogenesis. METHODS The HBx transgenic mice were produced by microinjecting the construct with X gene of HBV (subtype adr) DNA fragment into fertilized eggs derived from inbred C57 BL/6 strain; transgenic mice were identified by using Nested PCR; expression and phenotype of HBx gene were analyzed in liver from transgenic mice at the age of 8 weeks by RT PCR, pathologic examination and periodic acid schiff staining (PAS), respectively. RESULTS Five hundred and fourteen fertilized eggs of C57 BL/6 mice were microinjected with recombinant retroviral DNA fragment, and 368 survival eggs injected were transferred to the oviducts of 18 pseudopregnant recipient mice, 8 of them became pregnant and gave birth to 20 F1 offspring. Of 20 offsprings, four males and two females carried the hybrid gene (HBx gene). Four male mice were determined as founder, named X1, X5, X9 and X15. These founders were back crossed to set up F1 generations with other inbred C57BL/6 mice or transgenic littermates, respectively. Transmission of HBx gene in F1 offspring of X1, X5 and X9 except in X15 followed Mendelian rules. The expression of HBx mRNA was detected in liver of F1 offspring from the founder mice (X1 and X9), which showed vacuolation lesion and glycogen positive foci. CONCLUSION Transgenic mice harboring HBx gene were preliminarily established.

Detection of hyper-conserved regions in hepatitis B virus X gene potentially useful for gene therapy

AIM To detect hyper-conserved regions in the hepatitis B virus(HBV) X gene(HBX) 5' region that could be candidates for gene therapy.METHODS The study included 27 chronic hepatitis B treatmentnaive patients in various clinical stages(from chronic infection to cirrhosis and hepatocellular carcinoma, both HBeA g-negative and HBeA g-positive), and infected with HBV genotypes A-F and H. In a serum sample from each patient with viremia > 3.5 log IU/m L, the HBX 5' end region [nucleotide(nt) 1255-1611] was PCRamplified and submitted to next-generation sequencing(NGS). We assessed genotype variants by phylogenetic analysis, and evaluated conservation of this region by calculating the information content of each nucleotide position in a multiple alignment of all unique sequences(haplotypes) obtained by NGS. Conservation at the HBx protein amino acid(aa) level was also analyzed.RESULTS NGS yielded 1333069 sequences from the 27 samples, with a median of 4578 sequences/sample(2487-9279, IQR 2817). In 14/27 patients(51.8%), phylogenetic analysis of viral nucleotide haplotypes showed a complex mixture of genotypic variants. Analysis of the information content in the haplotype multiple alignments detected 2 hyper-conserved nucleotide regions, one in the HBX upstream non-coding region(nt 1255-1286) and the other in the 5' end coding region(nt 1519-1603). This last region coded for a conserved amino acid region(aa 63-76) that partially overlaps a Kunitz-like domain.CONCLUSION Two hyper-conserved regions detected in the HBX 5' end may be of value for targeted gene therapy, regardless of the patients' clinical stage or HBV genotype.

Upreguiation of human telomerase reverse transcriptase mRNA expression by in vitro transfection of hepatitis B virus X gene into human hepatocarcinoma and cholangiocarcinoma cells

AIM： To study the changes of human telomerase reverse transcriptase （hTERT） mRNA expression in human hepatocarcinoma cell lines （HepG2） and cholangiocarcinoma cell lines （QBC939） after HBx gene transfection and to illustrate the significance of transcriptional regulation of hTERT gene by HBx gene in the carcinogenesis. METHODS： HepG2 and QBC939 cell lines were cultured and co-transfected with eukaryotic expression vector containing the HBx coding region and cloning vector containing enhanced green fluorescent protein （EGFP） coding sequence using lipid-mediated gene transduction technique. Thirty-six hours after transfection, EGFP expression in cells was used as the indicator of successful transfection. Flow cytometry was performed to determine the transfection efficiency. Cells were harvested and total RNA was extracted using TRIzol reagent. The expression of hTERT mRNA in HepG2 and QBC939 cell lines was assayed by reverse transcriptionpolymerase chain reaction. The expression of HBx protein in both cell lines was detected by immunocytochemical staining and Western blotting. RESULTS： Flow cytometry showed that the transfection efficiency was 46.4% in HepG2 cells and 29.6% in QBC939 cells for both HBx gene expression vector and blank vector. The expression of hTERT mRNA was meaningfully increased in HepG2 and QBC939 cell lines when transfected with HBx gene expression vector compared to those transfected with OPTI-MEM medium and blank vector. Immunocytochemical staining and Western blotting revealed HBx protein expression in HepG2 and QBC939 cells only when transfected with HBx gene. CONCLUSION： HBx gene transfection can upregulate the transcriptional expression of hTERT mRNA. The transactivation of hTERT gene by HBx gene is a newfound mechanism for pathogenesis of hepatocarcinomas and cholangiocarcinomas after HBV infection. 2005 The WJG Press and Elsevier Inc. All rights reserved

Differentially expressed genes in hepatocellular carcinoma induced by woodchuck hepatitis B virus in mice

INTRODUCTIONHepatocellular carcinoma(HCC)is one of the major causes of death in the word.The mechanism of carcinogenesis is unknown,although it is widely accepted that HBV and HCV are clsely related to liver cancer[1-5[1-5].Previously,a variety of studies have described the differences in gene expression which distinguished tumor from nontumor[6-11].Cloning of the genes,especially the genes associated with HBV and HCV,is still very important to account for the development of liver cancer.

Characterization of hepatitis B virus X gene quasispecies complexity in mono-infection and hepatitis delta virus superinfection

Hepatitis delta virus(HDV) seems to strongly suppress hepatitis B virus(HBV)replication, although little is known about the mechanism of this interaction. Both these viruses show a dynamic distribution of mutants, resulting in viral quasispecies. Next-generation sequencing is a viable approach for analyzing the composition of these mutant spectra. As the regulatory hepatitis B X protein(HBx) is essential for HBV replication, determination of HBV X gene(HBX)quasispecies complexity in HBV/HDV infection compared to HBV monoinfection may provide information on the interactions between these two viruses.AIM To compare HBV quasispecies complexity in the HBX 5' region between chronic hepatitis delta(CHD) and chronic HBV mono-infected patients.METHODS Twenty-four untreated patients were included: 7/24(29.2%) with HBeAgnegative chronic HBV infection(CI, previously termed inactive carriers), 8/24(33.3%) with HBeAg-negative chronic hepatitis B(CHB) and 9/24(37.5%) with CHD. A serum sample from each patient was first tested for HBV DNA levels.The HBX 5' region [nucleotides(nt) 1255-1611] was then PCR-amplified for subsequent next-generation sequencing(MiSeq, Illumina, United States). HBV quasispecies complexity in the region analyzed was evaluated using incidencebased indices(number of haplotypes and number of mutations), abundancebased indices(Hill numbers of order 1 and 2), and functional indices(mutation frequency and nucleotide diversity). We also evaluated the pattern of nucleotide changes to investigate which of them could be the cause of the quasispecies complexity.RESULTS CHB patients showed higher median HBV-DNA levels [5.4 logIU/mL,interquartile range(IQR) 3.5-7.9] than CHD(3.4 logIU/mL, IQR 3-7.6)(P = n.s.)or CI(3.2 logIU/mL, IQR 2.3-3.5)(P < 0.01) patients. The incidence and abundance indices indicated that HBV quasispecies complexity was significantly greater in CI than CHB. A similar trend was observed in CHD patients, although only Hill numbers of order 2 showed statistically significant differences(CHB2.81, IQR 1.11-4.57 vs CHD 8.87, 6.56-11.18, P = 0.038). There were no significant differences in the functional indices, but CI and CHD patients also showed a trend towards greater complexity than CHB. No differences were found for any HBV quasispecies complexity indices between CHD and CI patients. G-to-A and C-to-T nucleotide changes, characteristic of APOBEC3 G, were higher in CHD and CI than in CHB in genotype A haplotypes, but not in genotype D. The proportion of nt G-to-A vs A-to-G changes and C-to-T vs T-to-C changes in genotype A and D haplotypes in CHD patients showed no significant differences. In CHB and CI the results of these comparisons were dependent on HBV genotype.CONCLUSION The lower-replication CHD and CI groups show a trend to higher quasispecies complexity than the higher-replication CHB group. The mechanisms associated with this greater complexity require elucidation.

Host cellular micro RNA involvement in the control of hepatitis B virus gene expression and replication

A large number of studies have demonstrated that the synergistic collaboration of a number of micro RNAs(mi RNAs), their growth factors and their downstream agents is required for the initiation and completion of pathogenesis in the liver. mi RNAs are thought to exert a profound effect on almost every aspect of liver biology and pathology. Accumulating evidence indicates that several mi RNAs are involved in the hepatitis B virus(HBV) life cycle and infectivity, in addition to HBVassociated liver diseases including fibrosis, cirrhosis and hepatocellular carcinoma(HCC). In turn, HBV can modulate the expression of several cellular mi RNAs, thus promoting a favorable environment for its replication and survival. In this review, we focused on the involvement of host cellular mi RNAs that are directly and indirectly associated with HBV RNA or HBV associated transcription factors. Exploring different facets of the interactions among mi RNA, HBV and HCV infections, and the carcinogenesis and progress of HCC, could facilitate the development of novel and effective treatment approaches for liver disease.

Possible mechanism for hepatitis B virus X gene to induce apoptosis of hepatocytes

AIM： To investigate the possible mechanism for HBV X gene to induce apoptosis of hepatocyte HL-7702 cells.METHODS： HBV X gene eukaryon expression vector pcDNA3-X was established and transfected into HL-7702 cells by lipid-mediated transfection, including transient and stable transfection. Positive clones were screened by incubating in the selective medium with 600 μg/mL G418 and named HL-7702/HBV-encoded X protein （HBx） cells. The expressions of Fas/FasL, Bax/Bcl-2, and c-myc mRNA were measured by semi-quantitative RT-PCR in HL-7702/HBx and control group, respectively.RESULTS： RT-PCR analysis confirmed that HBV X gene was transfected into HL-7702 cells successfully. By semiquantitative RT-PCR analysis, Bax and c-myc mRNA levels in HL-7702/HBx cells of transient transfection were significantly higher than those in control, FasL and c-myc mRNA levels in HL-7702/HBx cells of stable transfection were significantly higher than those in control, whereas the Bcl-2 mRNA levels in HL-7702/HBx cells of transient and stable transfection were significantly lower than thosein control.CONCLUSION： HBV X gene may promote the apoptosis of hepatocytes by regulating the expressions of Fas/FasL, Bax/Bcl-2, and c-myc gene in a dose-dependent manner.

Biological impact of hepatitis B virus X-hepatitis C virus core fusion gene on human hepatocytes

AIM： To investigate the biological impact of hepatitis B virus X- hepatitis C virus core （HBV X-HCV C） fusion gene on hepatoma cells.METHODS： The recombinant adenoviruses Ad- XC, Ad-X and Ad-C expressing HBV X-HCV C fusion gene, HBVX gene and HCV C gene were constructed, respectively. Hepatoma cells were infected with different recombinant adenoviruses. MTT, colony- forming experiment, FCM, TUNEL assay were performed to observe the biological impact of the HBV X-HCV C fusion aene on liver cells.RESULTS： MTT showed that the Ad-XC group cells grew faster than the other group cells. Colony-forming experiment showed that the colony-forming rate for the Ad-XC group cells was significantly higher than that for the other group cells. FCM analysis showed that Ad-XC/Ad-X/Ad-C infection enhanced the progression of G1→S phase in the HepG2 cell cycle. The apoptosis index of the Ad-XC, Ad-X, Ad-C group cells was significantly lower than that of the AdO and control group cells. Semi-quantitative RT-PCR showed that the expression level of c-myc was the highest in Ad- XC infected cells. Tumor formation was found at the injected site of mice inoculated with Ad-XC-infected LO2 cells, but not in control mice.CONCLUSION： Ad-XC, Ad-X and Ad-C facilitate the proliferation activity of HepG2 cells and inhibit their apoptosis in vitro. The effect of Ad-XC is significantly stronger than that of Ad-X and Ad-C. Up-regulation of c-myc may be one of the mechanisms underlying the synergism of HBVX and HCV C genes on hepatocarcinogenesis in athymic nude mice.

Exploitation of host clock gene machinery by hepatitis viruses B and C

Many aspects of cellular physiology display circadian(approximately 24-h)rhythms.Dysfunction of the circadian clock molecular circuitry is associated with human health derangements,including neurodegeneration,increased risk of cancer,cardiovascular diseases and the metabolic syndrome.Viruses triggering hepatitis depend tightly on the host cell synthesis machinery for their own replication,survival and spreading.Recent evidences support a link between the circadian clock circuitry and viruses’biological cycle within host cells.Currently,in vitro models for chronobiological studies of cells infected with viruses need to be implemented.The establishment of such in vitro models would be helpful to better understand the link between the clock gene machinery and viral replication/viral persistence in order to develop specifically targeted therapeutic regimens.Here we review the recent literature dealing with the interplay between hepatitis B and C viruses and clock genes.

The influence of hepatitis B virus X protein on the clock genes in liver cells and its significance

Objective： The aim of this study was to investigate the influence of hepatitis B virus X protein （HBx） on the clock genes in LO2 cells and its significance. Methods： A cell line LO2-HBx, Stably transfected with HBx gene, was established. The levels of mRNA and protein expression of CLOCK and BMAL1 were detected by real-time PCR and western blot. Resuits： The expression of CLOCK mRNA and protein were increased in cell line LO2-HBx （P 〈 0.05）, while the expression of BMAL1 mRNA and protein were decreased in cell line LO2-HBx （P 〈 0.05）. Conclusion： The expressions of core clock gene CLOCK and BMAL1 have been changed by HBx, which breaks down the previous circadian rhythm of liver cells. This maybe one of the reasons leads to the formation of liver cancer.

Identification of cellular genes showing differential expression associated with hepatitis B virus infection

AIM: To investigate the impact of hepatitis B virus (HBV) infection on cellular gene expression, by conducting both in vitro and in vivo studies. METHODS: Knockdown of HBV was targeted by stable expression of short hairpin RNA (shRNA) in huH-1 cells. Cellular gene expression was compared using a human 30K cDNA microarray in the cells and quantified by real-time reverse transcription-polymerase chain reaction (RT-PCR) (qRT-PCR) in the cells, hepatocellular carcinoma (HCC) and surrounding non-cancerous liver tissues (SL). RESULTS: The expressions of HBsAg and HBx protein were markedly suppressed in the cells and in HBx transgenic mouse liver, respectively, after introduction of shRNA. Of the 30K genes studied, 135 and 103 genes were identified as being down- and up-regulated, respectively, by at least twofold in the knockdown cells. Functional annotation revealed that 85 and 62 genes were classified into four up-regulated and five down-regulated functional categories, respectively. When gene expression levels were compared between HCC and SL, eight candidate genes that were confirmed to be up- or down-regulated in the knockdown cells by both microarray and qRT-PCR analyses were not expressed as expected from HBV reduction in HCC, but had similar expression patterns in HBV- and hepatitis C virus-associated cases. In contrast, among the eight genes, only APM2 was constantly repressed in HBV non-associated tissues irrespective of HCC or SL. CONCLUSION: The signature of cellular gene expression should provide new information regarding the pathophysiological mechanisms of persistent hepatitis and hepatocarcinogenesis that are associated with HBV infection.

Cloning and expression of the preS1 gene of hepatitis B virus in yeast cells

Objective: To investigate the complex functions of HBV preS1 protein, we constructed HBV preS1 gene expression vector and expressed it in yeast cells. Methods: Polymerase chain reaction (PCR) was per- formed to amplify the gene of HBV preS1 from the plasmid pCP10 containing the whole DNA fragment of HBV ayw subtype as template and the PCR prod- uct was cloned into the pGEM-T vector for sequen- cing. After being identified, the HBV preSl gene was cut from the pGEM-T vector by EcoR I and Pst I restriction enzymes, and cloned into yeast expres- sive plasmid pGBKT7 to constructe pGBKT7-preS1 recombinant expressive plasmid. This plasmid was transformed into yeast cell AH109 and expressed in it. The yeast protein was isolated and analyzed with sodium dodecyl suifate-polyacrylamide gel electro- phoresis(SDS-PAGE) and Western blotting. Results: The HBV preS1 gene was amplified success- fully and identified by DNA sequencing. The PCR products were coincided completely with the reported sequence. The digested fragments were cloned into the pGBKT7 vector and transformed into yeast cell AH109. The results of SDS-PAGE and Western blot- ting assay showed: (1) The HBV preS1 protein was expressed and existed in yeast cells; (2) The molecu- lar weight of the expression product was about 30 000 D. Conclusion: The HBV preS1 gene was successfully cloned and expressed in yeast cells.

Inhibition of Hepatitis B Virus Replication by Rheum palmatum L. Ethanol Extract in a Stable HBV-producing Cell Line

肝炎 B 病毒(HBV ) 感染是在世界上的一个严重健康问题。然而，仍然为 HBV 感染没有令人满意的治疗学的策略。到为有更高的功效和更少的副作用的新 anti-HBV 代理人的搜索，繁体中文药感冒 palmatum L 的禁止的活动。对 HBV 复制的乙醇摘录(RPE ) 在这研究被调查。量的即时聚合酶链反应(PCR ) 被采用在稳定的生产 HBV 房间线 HepAD38 对 HBV-DNA 复制分析 RPE 的禁止的活动；HBV 表面抗原(HBsAg ) 和 e 抗原(HBeAg ) 的表示层次被酶也决定在 RPE 处理以后的连接 immunosorbent 试金(ELISA ) 。RPE 能 dose-dependently 禁止 HBV-DNA 和 HBsAg 的生产。50% 抑制(IC50 ) 的集中在 209.63 点被计算， 252.53 渭 g /mL， respectivel y。然而，它对 HBeAg 表示的禁止的活动甚至在高集中是细微的。RPE 在 HepAD38 房间上有弱细胞毒素的效果(CC50 = 1 640 渭 g /mL ) 并且选择索引(SI ) 在 7.82 点被计算。与二 anthraquinone 衍生物 emodin 和 rhein 相比， RPE 显示出 anti-HBV 和更弱的 cytotoxicity 的更高的能力。那么感冒 palmatum L。可能拥有能有效地禁止 HBV-DNA 复制和 HBsAg 表示的另外的功能的代理人。他们改进功效并且减少的结构的活跃代理人，鉴定和修正的进一步的纯化 cytotoxicity 被要求。关键词肝炎 B 病毒(HBV )- 抗病毒 - 感冒 palmatum L.ethanol 摘录(RPE )- HepAD38 房间 CLC 数字 R373 同等地相应的作者。

Tea Polyphenols Exerts Anti-hepatitis B Virus Effects in a Stably HBV-transfected Cell Line

In this study, the anti-HBV effects of tea polyphenols （TP） were examined. After cells were exposed to TP for 3, 6, 9 days, amounts of HBsAg, HBeAg and HBV-DNA released into the supernatant of the cultured HepG2 2.2.15 cells were detected. TP, to some extent, inhibited the secretion of HBsAg and strongly suppressed the secretion of HBeAg in a dose-dependent （P〈0.01） and time-dependent manner, with 50% maximal inhibitory concentration （IC50） value being 7.34μg/mL on the 9th day, but the time-dependence was not significant （P=0.051）. Expression of HBV-DNA in the supernatant of the cell culture also was significantly decreased in a dose-dependent fashion （P〈0.01）. The ICS0 of TP in inhibiting HBV DNA was 2.54 pg/mL. It concluded that TP possessed potential anti-HBV effects and may be used as a treatment alternative for HBV infection.

Adenovirus-expressed preS2 antibody inhibits hepatitis B virus infection and hepatic carcinogenesis

AIM:To investigate the inhibitory effect of hepatitis B virus (HBV) preS2 antibody (preS2Ab) against HBV in-fection and HBV-associated hepatic carcinogenesis.METHODS:An adenoviral vector carrying the full-length light and heavy chains of the HBV preS2Ab gene,Ad315-preS2Ab,was constructed.Enzyme linked immunosorbent assay (ELISA) and Western blotting analyses were used to determine the preS2Ab expres-sion levels in vitro.Immunofluorescent techniques were used to examine the binding affinity between the expressed HBV preS2Ab and HBV-positive liver cells.ELISAs were also used to determine hepatitis B surface antigen (HBsAg) levels to assess the inhibitory effect of the preS2Ab against HBV infection in L02 cells.The inhibitory effect of preS2Ab against hepatic carcinogen-esis was studied with diethylnitrosamine (DEN)-induced hepatocellular carcinomas (HCCs) in HBV transgenic mice.RESULTS:The expression of HBV preS2Ab increased with increases in the multiplicity of infection (MOI) of Ad315-preS2Ab in L02 cells,with 350.87 ± 17.37 μg/L of preS2Ab when the MOI was 100 plaque forming units (pfu)/cell.The expressed preS2Abs could recog-nize liver cells from HBV transgenic mice.ELISA results showed that L02 cells expressing preS2Ab produced less HBsAg after treatment with the serum of HBV pa-tients than parental L02 cells expressing no preS2Ab.HBV transgenic mice treated with Ad315-preS2Ab had fewer and smaller cancerous nodes after induction with DEN than mice treated with a blank Ad315 vec-tor or untreated mice.Additionally,the administration of Ad315-preS2Ab could alleviate hepatic cirrhosis and decrease the serum levels of alanine transaminase and aspartate transaminase.CONCLUSION:Adenovirus-mediated HBV preS2Ab expression could inhibit HBV infection in L02 cells,and then inhibit DEN-induced hepatocellular carcinogenesis and protect hepatic function in HBV transgenic mice.

Identification and Characterization of Hepatitis B Virus Immune Escape Mutants in Kenya

Hepatitis B Virus (HBV) infections affect about 400 million people globally and cause about 1.4 million deaths annually. The virus displays high levels of genetic variations/mutations, some of which are immune escape mutants. The prevalence of HBV infection in Kenya is high at about 8%. This study aimed at identifying and characterizing HBV immune escape mutants in Kenya. From 547 HBV sequences available in Kenya in NCBI, and HBVdb databases in July 2021, 120 full sequences were retrieved. The S gene sequences at position 1-225, which included the “a” determinant region of the gene were analyzed using various bioinformatics tools such as Bioedit software, and Emboss Cons. The clinical significance was flagged from the search of peer-reviewed journals. Forty-six HBV-positive blood donor samples were obtained from the Kenya National Blood Transfusion Services without personal identifiers, DNA extracted, and sequenced targeting positions 1 to 520 of S genes. Mutations were similarly identified from seventeen sequences after cleaning and analysis. Out of 120 sequences that were extracted from databases and analyzed, 79 different mutations were identified. Fifteen of them were of clinical importance with an occurrence frequency of at least 5% were obtained. The majority (64.6%, n = 51), with S207N and A194V being most dominant, could result in immune escape and reduced HBsAg detection signals while 24.1% (n = 19) could result in immune escape/reduced HBsAg detection signals and high probability of hepatocellular carcinoma. Most likely to occur on the amino acids Alanine, Lysine, Serine, Asparagine, and Valine in decreasing order. The most dominant genotype was found to be Genotype A (N = 10), while four sequences were Genotype D. In contrast to the in-silico studies, the sequences from HBV samples from blood donors did not demonstrate the presence of S207N and A194V mutations and all the genotypes were type A1. Only two (13.3%) samples showed the same mutations of sK122R and sT143S for both in-silico analysis and actual sequenced samples. This study did not identify G145R mutation which is the commonest mutation within the HBsAg immunodominant “a” determinant that is associated with immune escape. The concordance of mutations in “a” determinant region of HBsAg gene among various studies is minimal. The study identified new mutations (sA194Y, sS207, sA194S, sS207I, sP46A, sA194T, sS207I, sP46R, and sT143P) that had not been published before. Four (20%) of the mutations were clinically significant. These included sS207R, sT143S, sC76F and sK122R.

Construction of a replication-competent hepatitis B virus vector carrying secreted luciferase transgene and establishment of new hepatitis B virus replication and expression cell lines

BACKGROUND Previously,we have successfully constructed replication-competent hepatitis B virus(HBV)vectors by uncoupling the P open reading frame(ORF)from the preC/C ORF to carefully design the transgene insertion site to overcome the compact organization of the HBV genome and maintain HBV replication competence.Consequently,the replication-competent HBV vectors carrying foreign genes,including pCH-BsdR,carrying blasticidin resistance gene(399 bp),and pCH-hrGFP,carrying humanized renilla green fluorescent protein gene(720 bp),were successfully obtained.However,the replication efficiency of the former is higher but it is tedious to use,while that of the latter is poor and cannot be quantified.Hence,we need to search for a new reporter gene that is convenient and quantifiable for further research.AIM To establish a helpful tool for intracellular HBV replication and anti-viral drugs screening studies.METHODS We utilized the replication-competent HBV viral vectors constructed by our laboratory,combined with the secreted luciferase reporter gene,to construct replication-competent HBV vectors expressing the reporter gene secretory Nanoluc Luciferase(SecNluc).HepG2.TA2-7 cells were transfected with this vector to obtain cell lines with stably secreted HBV particles carrying secNluc reporter gene.RESULTS The replication-competent HBV vector carrying the SecNluc reporter gene pCHsNLuc could produce all major viral RNAs and a full set of envelope proteins and achieve high-level secreted luciferase expression.HBV replication intermediates could be produced from this vector.Via transfection with pTRE-sNLuc and selection by hygromycin,we obtained isolated cell clones,named HBV-NLuc-35 cells,which could secrete secNLuc recombinant viruses,and were sensitive to existing anti-HBV drugs.Using differentiated HepaRG cells,it was verified that recombinant HBV possessed infectivity.CONCLUSION Our research demonstrated that a replication-competent HBV vector carrying a secreted luciferase transgene possesses replication and expression ability,and the established HBV replication and expression cell lines could stably secrete viral particles carrying secNluc reporter gene.More importantly,the cell line and the secreted recombinant viral particles could be used to trace HBV replication or infection.